The present invention relates in general to oligonucleotides labeled with a plurality of spectrally identical or similar dyes and optionally one or more quencher dyes, as well as methods of creating labeled oligonucleotides and various uses of the labeled oligonucleotides as primers or probes for highly sensitive nucleic acid detection, including real-time polymerase chain reaction (PCR).
Nucleic acid polymers such as DNA and RNA are essential to the transmission of genetic information from one generation to the next and in the routine functioning of all living organisms. Accordingly these molecules are the object of intense study and a number of techniques have been developed to study of these molecules. These methods include but are not limited to methods for identifying the presence of a specific polynucleotide sequences in a given sample and methods designed to measure the number of specific nucleic acid molecules originally present in a given sample.
Practical uses for these techniques include identifying specific species and relationships between various species based upon similarities in oligonucleotide sequences. Other uses include diagnosing disease by identifying specific sequences in a given sample as indicative of a given pathology. Still other uses, too numerous to mention, include identifying individuals with a predisposition for developing a specific pathology as well as assessing the efficacy of proposed treatment regimes based on the presence of specific polynucleotides in a given patient's genome.
One of the most widely used and powerful techniques for the study and manipulation of oligonucleotides is the polymerase chain reaction (PCR). PCR is a primer extension reaction that provides a method for amplifying specific nucleic acids in vitro. This technique was first described in 1987. PCR can produce million fold copies of a DNA template in a single enzymatic reaction mixture within a matter of hours, enabling researchers to determine the size and sequence of target DNA. This DNA amplification technique has been widely used for cloning and other molecular biological manipulations. Further discussion of PCR is provided in Mullis et al., Methods Enzymol. (1987); and Saiki et al., Science (1985).
In PCR the particular stretch of DNA to be amplified is referred to as the ‘target sequence’. The target sequence is replicated by first binding a complimentary ‘primer’ to a single stranded portion of the target polynucleotide. One PCR based technique that is particularly useful is Quantitative PCR (qPCR). Briefly, the mechanism of qPCR is based on the fact that PCR amplifies a target DNA in an exponential manner. By running a PCR reaction and measuring the total number of DNA copies at given points during the course of the amplification reaction, one can retroactively calculate the amount of starting DNA material.
Various methods have been developed for determining the amount of PCR product made without having to stop the PCR run or even to sample the reaction during a given PCR run. One such method follows the course of the PCR run in real time by measuring the amount of product at each cycle of DNA synthesis. This process is referred to as real time PCR (RT-PCR). Because of its great sensitivity and because measurements can be made with the sample still in the PCR thermocylcer, various fluorescence-based assays that monitor the formation of PCR products have been developed. A number of instruments and methods have been developed for real-time PCR (RT-PCR). A real-time PCR instrument is typically a fluorometer built upon a thermocycler. Commercially available real-time PCR instruments include Prism7700 by ABI, LightCycler by Roche, Opticon by MJ Research, iCycler IQ by BioRad, and MX4000 by Stratagene.
An oligonucleotide used to identify a given sequence of nucleic acid by hybridizing to it, but that does not serve to amplify the sequence may be referred to as a ‘probe’. Probes also find utility in PCR reactions where they are used to signal polynucleotide amplification.
Given the importance of oligonucleotide and the myriad of ways in which these molecules can impact human, animal and plant life there is a need for ever more efficient methods for the study and manipulation of oligonucleotide. Including new techniques for efficiently producing labeled oligonucleotide. One object of the present invention is to provide labeled oligonucleotide and efficient methods for making and using the same.